Laccase Protocols

From OpenWetWare

Revision as of 22:57, 10 October 2011 by Michael A. Speer (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Back to Protocols

Laccase Protocols

Contents

Introduction

This protocols are used to screent for and assay laccase activity.

ABTS Plate Screen

Materials

  • ABTS 20mM (10mg/mL) stock solution
  • CuSO4 100mM (25mg/mL) stock solution

Procedure

1. Make a liter of your favorite agar growth medium and autoclave.
2. After cooling to 65°C add:

  • 10ml/ABTS (0.2 mM)
  • 1mL CuSO4

3. Swirl to mix and pour plates.
4. Once plates are hard:

  • Drop 5μL of overnight culture onto the plate (when this dries it will form a little circle).
  • Culture plate 24-48 hours
  • Observe green halos around ABTS oxidizing cultures.

Notes

References

  • Soden et al. (2002) Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host. Microbiology 148 (2002), 4003-4014

Bromophenol Blue Plate Screen

Materials

  • Agar medium of your choice.
  • Bromophenol blue

Procedure

  1. Supplement the agar medium with 0.2g/L bromophenol blue.
  2. Autoclave and pour plates.
  3. Culture microorganism on plates
  4. Observe clear halos around laccase producting colonies.

Notes

References

  • Tekere et al. 2001. Growth, dye degradation and ligninolytic activity studies on Zimbabwean white rot fungi. Enzyme and Microbial Technology 28 (2001) 420–426

DMP Assay

Materials

  • DMP (2,6-dimethoxyphenol)
  • DMS (2,2 dimethyl succinate)
    • Can also substitute with sodium malonate

Procedure

1. Prepare substrate solution:

  • 4mM DMP (620mg/L)
  • 200mM DMS (29.2g/L)

2. Centrifuge 1ml of overnight culture.
3. Place 750μL of cleared supernatant in a microcentrifuge tube.
4. Add 250μL of substrate solution.
5. Incubate for 10 minutes.
6. Measure absorbance at 468nm every minute.

  • An increae in absorbance is positive for laccase activity.
  • Extinction coefficienct = = 49.6 AU/mM*cm

Notes

References

Guiacol Screen

Materials

  • Guiacol (or gum guaiac)
  • Ethanol
  • Agar medium

Plate screen Procedure

1. Add 0.01% guiaiacol to agar medium (100μL/L).
2. Autoclave and pour plates.
3. Culture microorganisms on plates.
4. Observe orange/brown halos around laccase positive colonies.

Drop Screen Procedure

1. Dissolve either 17mg gum guaiac or 13mg guaiacol into 1ml of 100% ethanol.

  • If your guaiacol is in liquid form then add 12μL to 1ml ethanol.

2. Drop one drops of this solution on a large colony (2 days old for bacteria).
3. Wait four hours and a laccase positive colony should turn orange/brown.
4. Bacteria don't usually like ethanol, so now they're probably dead.

Notes

  • These resutls can be difficult to observe if the medium is already brownish.

References

  • Kiiskinen et al. 2004. Screening for novel laccase-producing microbes. Journal of Applied Microbiology 2004, 97, 640–646
  • Lopez et al. 2005. Decolorization of industrial dyes by ligninolytic microorganisms isolated from composting environment. Enzyme and Microbial Technology 40 (2006) 42–45

Lignin Plate Screen

Materials

  • Plates containing:
    • 20g/L agar
    • 5g/L glucose
    • 5g/L ammonium tartrate
    • 1g/L malt extract
    • 0.5g/L MgSO4 (heptahydrate)
    • 0.1g/L NaCl
    • 0.01g/L CaCl
    • 0.01g/L FeCl3
    • 0.01g/L kraft alkaline lignin
    • 1mg/L thiamine
  • Ferric Chloride Solution
    • 10g/L FeCl3
  • Potassium Ferric Cyanide Solution
    • 10g/L K3[Fe(CN)6]

Procedure

  1. Streak bacteria onto the plates and incubate for 5 days.
  2. Mix equal parts Ferric Chloride Solution and Potassium Ferric Cyanide Solution.
  3. Flood plates with this wash solution.
  4. The was solution will stain the lignin green. Non-green halos around colonies indicate lignin degradation.

Notes

  • Some protocols bump up the lignin concentration to 0.25g/L.

References

  • Sundman and Nase. 1974. A simple plate test for direct visualization for biological lignin degradation. Papper och Tra 2
  • Tekere et al. 2001. Growth, dye degradation and ligninolytic activity studies on Zimbabwean white rot fungi. Enzyme and Microbial Technology 28 (2001) 420–426


Back to Protocols

Personal tools